KR101498665B1 - Pharmaceutical composition and functional food for prevention or treatment of diabetic complications or angioedema comprising natural extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating diabetic complications or angioedema comprising, as an active ingredient, an Ivy leaf (Songak) extract and a Rhodiola mixture extract. More particularly, And a pharmaceutical composition for preventing or treating diabetic complications or angioedema which additionally contains Hojangun, Rhubarb, Changgeun, Ginkgo leaf, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe And a health functional food composition.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition and a functional food composition for preventing or treating diabetic complications or angioedema including natural extracts and a dietary supplement for treating diabetic complications or angioedema comprising natural extract,

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating diabetic complications or angioedema including an ivy leaf and a mixture of phytoplankton extract as an active ingredient. More particularly, The present invention relates to a pharmaceutical composition and a health functional food composition for the prevention or treatment of diabetic complications or angioedema which additionally contains an effective ingredient such as a diabetic rheumatoid arthritis gangrene, a ginkgo leaf, a lucifer, blueberry, bilberry, bokbunja, licorice, grape seed or aloe extract .

Diabetes mellitus is one of the most important adult diseases in the world. In recent years, with the rapid economic growth, the prevalence of diabetes in Korea has reached 10%, now more than 240 million people worldwide, and in 2025, And 60% of them will develop in Asia, according to the American Medical Association (JAMA) in 2009. In particular, the onset of diabetes was elicited by the elderly, and the prolongation of life span led to complications.

As the duration of diabetes mellitus increases, diverse complications of the system are accompanied by cardiovascular disease, diabetic nephropathy, diabetic neuropathy and diabetic retinopathy. Diabetic retinopathy (DR) occurs in more than 60% of diabetic patients within 10 years of diabetes diagnosis and more than 90% in 20 years. In all age groups, the rate of diabetic complications increased, rather than diabetes. In particular, the rate of peripheral circulatory disorder complications in patients aged 40-50 was 6.5 times higher than that in diabetic patients and 2.2 times higher in patients with diabetic retinopathy. Peripheral circulatory disorder The cost of diabetic complications increased from 54 billion won (KRW 80.7 billion in 2006) to 153 billion KRW in 2010 (54 million KRW in KRW, from KRW 32.7 billion in diabetic retinopathy) (Aug. 29, 2011). This is because the average life expectancy is increasing and the number of older patients with diabetes is increasing. This indicates how important it is to take precautions to prevent diabetic patients from developing diabetic complications.

Among diabetic complications, diabetic retinopathy is one of the microangiopathies of diabetes mellitus, which is characterized by changes in retinal vascular permeability, vascular occlusion, ischemia, neovascularization, and fibrovascular proliferation fibrovascular proliferation).

Diabetic retinopathy is the largest cause of acquired blindness in the United States, with 12,000 to 24,000 cases of blindness annually in the United States. In the United States, the prevalence of diabetic retinopathy is estimated to be about 40% of diabetic patients and about 8% is reported to be a serious condition that can lead to blindness.

Although there is a growing need for early detection and progression of diabetic retinopathy and early treatment of high-risk patients, the pathogenesis remains to be elucidated. It is not supported, and effective treatment is limited.

Also, in a morbid condition, the blood vessels and the lymph vessels are slackened, causing abnormalities in the function, and the tissue fluid and blood become stagnant. When this symptom is present in the vein, it appears in the varicose veins and lymphatic vessels, causing lymphatic edema. Histologically, varices appear in the lower extremities. This results in dyspnea, pain, sensory abnormalities, and night pain. However, there are no effective remedies for treating or improving the condition, and antioxidant medicines have been administered. If these symptoms appear in the blood vessels of the retina, macular edema and macular degeneration may be induced.

Macular is the nerve tissue in the middle of the retina, which is affected by chronic diabetes and bloodstream edema. That is, changes in drusen, retinal pigment epithelial atrophy, choroidal neovascua (rization), and the like result in the development of macular degeneration. Protein, red blood cells, etc. from retinal capillaries escape to the retina, and macular edema and macular degeneration occur, resulting in visual impairment (Diabetes Study 4, revised edition, Korea Diabetes Society, 2011, 577).

There is no effective treatment for macular degeneration. Up to now, only expensive antivascular endothelial growth factor therapy has been available. In addition, there is a local laser treatment, but it is not cured, and even if it is improved, there is still a problem that an uncomfortable visual acuity must be paid.

In addition, diabetic neuropathy is the most common complication of diabetes, occurring in more than 80% of diabetic patients. Neurological symptoms are caused by accumulation of metabolic byproducts, loss of myelin region of nerve cells, and changes in microvessels. Treatment of neurosis is generally not easy, and prevention through blood glucose control and relief of symptoms such as pain are being performed. Peripheral vascular disease due to diabetes is a disease found in 45% of patients after a period of about 20 years. It is accompanied by vascular stiffness, blood circulation disorder is a common symptom, and complete healing is known to be difficult. For example, a foot ulcer is a diabetic complication that results in a deadly result of limb amputation.

On the other hand, the production of the final glycation endproduct is known to be one of the typical factors causing diabetic complications as described above. Nonenzymatic glycation of protein is a process in which advanced glycation endproducts (AGEs) are formed by the condensation reaction (milliard reaction) of amino acid groups such as lysine residues of proteins and reducing sugars without enzymatic action .

Unlike the reversible amido type early saccharide carbohydrate, the final glycation end product is irreversible reaction product. Once formed, it is not degraded even when blood glucose is restored to normal, but accumulates in the tissue during the protein survival period, Diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, diabetic heart disease, diabetic osteoporosis, or diabetic atherosclerosis (Vinson, JA et al. al., 1996, J. Nutritional Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. J. Biochem., 210: 729-739).

Under the above background, the inventors of the present invention have found that a composition comprising an extract of Ivivifolia and Rhodiola as an active ingredient, or a composition comprising the extract of Ivy leaf and Rhodiola extract in combination with a composition comprising Hojangun, Rhubarb, Ganoderma Luciflora, Ginkgo biloba, Cucumber, Blueberry, Bilberry, , Grape seed or aloe extract is added to the composition to inhibit the formation of final glycation products, inhibit vascular endothelial growth factor formation, inhibit retinal blood barrier collapse, improve nerve conduction, and improve nephropathy, The present inventors completed the present invention by confirming that it is effective for preventing or treating angioedema.

It is an object of the present invention to provide a pharmaceutical composition and a health functional food composition containing, as an active ingredient, an ivy leaf and a mixture of Rhodiola extract capable of preventing or treating diabetic complications or angioedema.

 In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications or angioedema comprising the mixed extract of ivy leaves and Rhodiola as an active ingredient.

In addition, the composition of the present invention may further comprise an extract of Hojangun, Rhubarb, Puerariae, Ginkgo biloba, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe.

As used herein, the term "mixed extract of Ivy leaf and Rhodiola" means an extract obtained by extracting a mixture of Ivy leaf extract and Rhodiola extract or a mixture of Ivy leaf and Rhodiola.

As used herein, the term "Ivy leaf" refers to a leaf of Ivy that is a plant of the evergreen vine plant that grows on a stalk from other branches and is called Hedera rhombea in Korea. Leaves are 3 ~ 6 ㎝ in length, shiny and dark green. Leaves were used in ancient times as an addiction treatment, and are known to have the effect of relieving the pain of neuralgia, rheumatism and sciatica. The ivy leaf extract of the present invention is preferably produced by extracting ivy leaves with water, methanol, ethanol, butanol or a mixed solvent thereof, but is not limited thereto.

The term " Coptis chinensis " as used in the present invention means a perennial herbaceous plant belonging to the family Ranunculaceae. In the oriental herb, the roots of Rhododendron and Rhododendron are picked and dried in the sun. It has been used as a remedy for dyspepsia, gastritis, enteritis, abdominal pain, vomiting, dysentery, heart palpitations, burns, mental anxiety, sore throat, hematochezia, nosebleeds, The extract of Rhododendron japonica of the present invention is preferably produced by extracting Rhodiola with water, methanol, ethanol, butanol or a mixed solvent thereof, but is not limited thereto.

The term " Polygonun " cuneata ) "refers to the perennial plant of Pseudomonas spp., which is used as mitigation diuretic and general economy, and is used as sedative in the private sector.

As used herein, the term " Rheum palmatum "refers to perennial herbaceous perennials of the dicotyledonous plants of the dicotyledonous plant, and is used as an anti-inflammatory agent in the herbal medicine. When a small amount of the palmatum is used, it exhibits a dryness effect. In a large amount, It is good for constipation and indigestion, and it is also used for burns in civilian medicine.

As used herein, the term " Puerariae "refers to the root of Pueraria thynbergiana , a perennial copy belonging to the bean family . In China, Pueraria lobata or Pueraria thomsonii , Piglets are used for indigestion, headache, anemia, dysentery, abdominal pain, dryness, colds, vomiting and hemorrhaging of wives.

As used herein, the term "Ginkgo biloba (Ginkgo leaf "refers to the leaves of Ginkgo biloba , a deciduous tree of the ginkgo tree.

The term " killer " as used in the present invention refers to an annual herbaceous plant belonging to the soybean family called Cassia Tora L. ), and Cassia obtusifolia , and it is also called Yangbang, Embossed, Horseshoe, Horseradish, Mungbean, and Mungo. Blood pressure lowering, diuretic, transmission, uterine contraction and skin fungus suppression, and cholesterol hyperactivity.

As used herein, the term " Vaccinium spp., Blueberry "refers to a shrub of the azalea rhododendron of the dicotyledonous plant . The term " blueberry " .

The term " Bilberry "as used in the present invention means fruit of Vaccinium vitis-idaea , an evergreen fruit of the azalea spp ., And has functions of protecting eyesight, decreasing cholesterol, and preventing constipation.

The term " bokbunja " as used in the present invention means that the fruit of Rubus coreanus is dried, and has anti-inflammatory action, antioxidative action and anti-Helicobacter pylori action.

As used herein, the term " licorice "refers to licorice ( Glycyrrhiza uralensis , Glycyrrhiza glabra ) or other roots and stem. It is effective for detoxification, hepatitis, urticaria, dermatitis, eczema, etc., and has Jinhae genome, muscle relaxation, diuretic action, anti-inflammatory action and peptic ulcer inhibiting action.

Term "grape seed" used in the present invention vine (Vitis vinifera ), which means the seed of grape, which is effective for restoring fatigue and antivirus.

The term " Aloe "as used in the present invention means a perennial plant belonging to the genus Aloe with a lily, and has functions of enhancing immunity, gastrointestinal protection, promotion of blood circulation and the like.

The extract of the present invention of the present invention can be used as a herb extract or a herb extract in the form of a herb extract or a herb extract. Or aloe with water, methanol, ethanol, butanol or a mixed solvent thereof, but is not limited thereto.

The composition of the present invention is preferably prepared by mixing the above extract of Ivy leaf and Rhodiola extract at a ratio of 10: 1 to 1:10, or extracting the mixture of Ivy leaf and Rhodiola at a ratio of 10: 1 to 1:10, It is not.

In addition to the ivy leaf extract and Rhodiola extract of the present invention, it is also preferable that the composition further comprises metformin. When metformin is added, the effect of the composition of the present invention is remarkably enhanced. Metformin is preferably added in an amount of 1 to 7 times the total weight of the mixed extract of Ivy leaf and Rhodiola.

The extraction may be performed by room temperature extraction, hot water extraction, cold extraction, reflux cooling, ultrasonic extraction, supercritical or steam extraction, but is not limited thereto.

In addition, the ivy leaf extract of the present invention may include a fraction of ivy leaf extract.

The term "fraction " used in the present invention means a specific component obtained by suspending an ivy leaf extract in water and sequentially fractionating it with a solvent having a different polarity. The solvent may be ethyl acetate, n-butanol or water.

Specifically, the fraction of the ivy leaf extract may be one obtained by suspending an ivy leaf ethanol extract in water and then fractionating it with ethyl acetate. The fraction is suspended in water and then fractionated with ethyl acetate. The water fraction is fractionated with butanol Fractions or water fractions.

Also, it may be a butanol fraction or a water fraction obtained by suspending an ethanol extract of ivy leaves in water and then fractionating with butanol.

The term "diabetic complication" used in the present invention means a symptom caused when the diabetes persists for a long period of time. The diabetic complications are different from the criteria for the onset of diabetes and the criteria for judgment, and diabetic complications are used separately from the diabetic drugs. Diabetic complication diseases are typically diabetic peripheral vascular diseases such as diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, coronary artery disease, peripheral arterial disease, and cerebrovascular disease. Examples of angioedema include varicose veins, macular edema, or macular lesions.

In the present invention, it is preferable that the extract is a mixture of Ivy leaf and Rhodiolite mixed extract or Ivy leaf and Rhodiola extract in combination with Hojangun, Rhododendron, Puerariae, Ginkgo biloba, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe extract It was confirmed that the composition is effective for preventing or treating diabetic complications or angioedema. According to one embodiment of the present invention, the mixed composition prepared by adding grape seed, ginkgo leaf, gangrene, sorghum, rhubarb, or bilberry extract to the mixed extract of Ivy leaf extract and Rhodiola extract is 2 to 10 Fold inhibition effect on the final glycation end product formation. In addition, the above-mentioned mixed extract of ivy leaves and Rhodiola showed inhibition of blood retinal barrier damage, inhibition of vascular endothelial growth factor formation, inhibition of non-collapsing capillary blood vessels, improvement of motor nerve conduction, and improvement of nephropathy, thereby preventing diabetic complications or angioedema And the therapeutic efficacy was confirmed.

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients.

The composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dose of the mixed extract of ivy leaves and Rhodiola is preferably 1 mg / kg to 1500 mg / kg, and may be administered once to several times per day as needed.

The present invention also provides a health functional food composition for preventing or ameliorating diabetic complications or angioedema comprising a mixed extract of ivy leaves and Rhodiola as an active ingredient.

In addition, the composition of the present invention may further comprise an extract of Hojangun, Rhubarb, Puerariae, Ginkgo biloba, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe extract in the mixed extract of Ivy leaf and Rhizome.

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" is not limited to the corresponding items in general rules and general test methods approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

At this time, the content of the ivy leaf extract and the Huanghui extract according to the present invention, which are added to foods containing beverages in the process of manufacturing a health functional food, can be appropriately increased or decreased, By weight to 15% by weight.

The present invention is a natural extract effective for prevention or treatment of diabetic complications or angioedema, and is useful as a pharmaceutical composition for preventing or treating diabetic complications or angioedema, as well as a health functional food.

FIG. 1 is a schematic view illustrating a process for producing a sesame extract and a sesame extract fraction according to an embodiment of the present invention.
FIG. 2 is a graph showing the fluorescence emission caused by damage to the retinal barrier of blood vessels in an STZ-diabetic-induced animal model by administration of COPH (ivy leaf extract + Rhodiola extract) according to an embodiment of the present invention; and (b) The results of the analysis are shown.
FIG. 3 is a graph showing the results of the analysis of (a) proteinuria, (b) final saccharide product urine output (AGEs), and (c) antioxidant activity of STZ-diabetic induced animal model by COPH (ivy leaf extract + ) 8-OHdG Quantitative analysis of urine output.
FIG. 4 is a graph showing the effect of the COPH extract (extract of Ivy leaf + Rhodiola) or COPH + MET (ivy leaf extract + Rhodiola extract + metformin) on the retinal vascular permeability of a fluorescent substance in an animal model induced by diabetes according to an embodiment of the present invention The results of the inhibition analysis are shown.
FIG. 5 is a graph showing the effect of serum albumin on retinal tissues of an animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) according to an embodiment of the present invention The results of inhibition of extracorporeal efflux phenomenon are shown.
FIG. 6 is a graph showing the distribution of occludin in the animal model of diabetes induced by the administration of COPH (ivory leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) according to an embodiment of the present invention Inhibition assay.
FIG. 7 is a graph showing the results of analysis of substrate protein degradation in retinal tissues of an animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) according to an embodiment of the present invention Enzyme (MMP2) expression inhibition assay.
FIG. 8 is a graph showing the results of a comparison between the final glycation end product in the retinal blood vessels of an animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) (A) fluorescence microscopy and (b) ELISA quantitative analysis results.
FIG. 9 is a graph illustrating the effect of the COPH (extract of Ivy leaf + Rhododendron) or COPH + MET (ivy leaf extract + Rhodiola extract + metformin) on the retinal vascular endothelial growth factor (A) fluorescence microscope and (b) ELISA quantitative analysis results.
FIG. 10 is a graph showing the results of non-erosive retinal capillary formation of an animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) according to an embodiment of the present invention (a) fluorescence microscopy and (b) ELISA quantitative analysis results.
FIG. 11 is a graph showing the results of inhibition of retinal nerve cell damage in an animal model induced by diabetes by administration of COPH (extract of Ivy leaf + Rhodiola extract) or COPH + MET (ivy leaf extract + Rhodiola extract + metformin) according to an embodiment of the present invention .
FIG. 12 is a graph showing the results of the production of reactive oxygen species in retinal tissues of an animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) or COPH + MET (ivy leaf extract + HuangYin extract + metformin) according to an embodiment of the present invention Inhibition and active nitric oxide inhibition assay.
FIG. 13 shows the qualitative analysis (a) and the quantitative analysis (a) of the motility nerve conduction (MNCV) of the animal model induced by diabetes by administration of COPH (ivy leaf extract + HuangYin extract) according to an embodiment of the present invention will be.
FIG. 14 is a graph showing the results of a comparison between a sesame extract (HR), an ethyl acetate fraction (EA), a butanol fraction (Bu) and a water fraction (W) (A) Fluorescent microscope and (b) analysis of changes in blood vessel thickness (Here, ### means p <0.001 as compared with the normal group, and *** denotes hyperglycemia induction P < 0.001 for the group).
FIG. 15 is a graph showing the results of a comparison between the results of the visual field analysis of the vitreous retinal blood vessels in the zebrafish model by the treatment of the sona extract (HR), the sesame root extract (Hojang muscle) and the sesame extract and the Hojokgang extract mixed composition (HR + (A) Fluorescence microscope and (b) blood vessel thickness change analysis results. Here, ### means p <0.001 for the normal group, *** represents p <0.001 for the hyperglycemic group .
FIG. 16 is a graph illustrating the effect of COPH (extract of Ivy leaf + Rhodiola extract) on blood retinal barrier damage in a VEGF-induced animal model according to an embodiment of the present invention, The results are shown in Fig.
FIG. 17 is a graph showing the effect of COPH (extract of Ivy leaf + Rhodiola extract) administered according to one embodiment of the present invention on the retinal blood retinal barrier The results are shown in Fig.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  One: Ivy leaves  The extract and goldthread  Preparation of compositions containing extracts

After adding 30% (v / v) aqueous ethanol solution to the dried ivy leaves (300 g), the mixture was subjected to primary reflux cooling and filtration. The filtrate was collected by filtration, and 30% ethanol was added to the remaining ivy leaf residue. The filtrate was combined with the filtrate obtained in the first extraction and concentrated under reduced pressure. Water was added thereto again to concentrate azeotropically and then dried to obtain an extract of Ivy leaf (Songak).

Further, 50% (v / v) ethanol aqueous solution was added to the dry fermentation (300 g), followed by primary reflux cooling extraction, filtration, collecting the filtrate, adding 50% ethanol again to the remaining residue, Was combined with the filtrate obtained in the first extraction and concentrated under reduced pressure. After water was added to suspend it, water saturated butanol was added to collect only the butanol fraction and concentrated under reduced pressure. Water was added thereto, followed by azeotrope concentration, followed by lyophilization to obtain Rhodiola extract.

A sample of COPH was prepared by mixing Ivo leaf (Songak) extract and Rhodiola extract obtained by the above method in a weight ratio of 3: 1.

Example  2: Preparation of mixed composition containing natural herbal extract

15 grape samples were prepared by mixing Grape Seed, Ginkgo Leaf, Puerariae Radix, Radish Killer, Ryegrass or Raspberry extract or Bilberry extract in a certain ratio to the mixture of Ivy leaf (Songak) extract and Rhodiola extract (COPH) prepared in Example 1 Respectively.

1) Preparation of mixed composition containing ivy leaf extract, Huanglian extract and grape seed extract

The dried grape seed extract was purchased from Enterrim ^® of Hanlim Pharmaceuticals. The obtained grape seed extract was mixed with 1: 1, 1: 2, or 1: 3 weight ratio of COPH and 1: 1 of COPH and 1: 3 of a mixture of Ivy leaf extract and Rhodiola extract prepared in Example 1, , COPH + grape seed 2 (2: 1) and COPH + grape seed 3 (3: 1).

2) Preparation of mixed composition containing ivy leaf extract, Rhodiola extract and Ginkgo biloba extract

Ginkgo biloba extract has been used to buy a group of annexin-F ^® SK Chemicals. The obtained Ginkgo biloba extract was mixed with the Ivy leaf extract and the Rhodiola extract composition (COPH) prepared in Example 1 at a weight ratio of 1: 3 to prepare a sample COPH + ginkgo leaf (3: 1).

3) Preparation of mixed composition containing ivy leaf extract, Huanglian extract and Puerariae Radix extract

After adding 80% (v / v) ethanol aqueous solution to the dry brown legs, first extraction, filtration, collecting the filtrate, adding 80% ethanol again to the remaining residue of the purple residue, and then extracting the filtrate by the second extraction, And concentrated under reduced pressure. The obtained Puerariae radix extracts were mixed with 1: 1 or 1: 3 weight ratio of the extract of Ivy leaf extract and Rhodiola extract prepared in Example 1 at a weight ratio of 1: 1 or 1: 3 to prepare samples COPH + Puerariae 1 (1: 1) and COPH + Puerariae 2 : 1).

4) Preparation of mixed composition containing ivy leaf extract, Rhodiola extract and Cassiae extract

After adding 80% (v / v) aqueous ethanol solution to the desiccant, the filtrate was collected by filtration, and the remaining filtrate was subjected to second extraction after adding 80% ethanol to the remaining residue. And concentrated under reduced pressure. The obtained Cassiae Radix extracts were mixed with 1: 1, 1: 2 or 1: 3 weight ratio of COPH + 1: 1 (1: 1), COPH + Criterion 2 (2: 1) and COPH + Criterion 3 (3: 1) were prepared.

5) Preparation of a mixed composition containing ivy leaf extract, Rhodiola extract and Rhodiola extract

After adding 40% (v / v) aqueous ethanol solution to the dried rhubarb, it was subjected to the first reflux cooling extraction and filtration. The filtrate was collected, and the remaining rhubarb residue was added with 40% ethanol again and subjected to the second reflux cooling extraction. The combined filtrate was concentrated under reduced pressure. The obtained rhubarb extracts were mixed with 1: 1, 1: 2 or 1: 3 weight ratio of COPH + 1: 1 (1: 1), COPH + Rhubarb 2 (1: 2) and COPH + rhubarb 3 (1: 3).

6) Preparation of a mixed composition containing ivy leaf extract, Huaxia extract and Bilberry extract

Bilberry extract is dried tagen international drug was used to purchase the F ^®. The purchased Bilberry extract was mixed with the mixture of Ivy leaf extract and Rhodiola extract prepared in Example 1 at a weight ratio of 1: 1, 1: 2, 1: 3 or 3: 1 to prepare a sample COPH + bilberry 1 1: 1), COPH + bilberry 2 (2: 1), COPH + bilberry 3 (3: 1) and COPH + grape seed 4 (1: 3).

Example  3: Ivy leaves (Songak) extract, goldthread  Preparation of compositions comprising extracts and metformin

350 parts by weight of metformin was added to 50 parts by weight of the Ivy leaf (Songak) extract and Rhodiola extract mixture (COPH) prepared in Example 1 to prepare a sample COPH + MET.

Example  4: Songa extract and Songa extract Fraction  Produce

The fractions of the Songa extract and the Songa extract were prepared according to the following method, and a schematic diagram of the production process is shown in Fig.

1) Preparation of Songmae extract (HR)

Shredded shrubs were cut into pieces and then repeatedly extracted with 1 liter of 80% ethanol, concentrated under reduced pressure at 40 ° C and dried under reduced pressure to obtain an ivy leaf ethanol extract (HR).

2) Preparation of ethylacetate fraction (EA) of Songmae extract

The Songmae ethanol extract prepared in Example 4-1) was suspended in water, and then ethyl acetate was added thereto for systematic separation. The separated ethyl acetate layer was concentrated under reduced pressure to obtain an ethylacetate fraction (EA) of Songmae ethanol extract.

3) Preparation of butanol fraction (Bu) of Songmae extract

The remaining water layer in Example 4-2) was mixed with n-butanol to separate the butanol dissolution layer. The separated butanol layer was concentrated under reduced pressure to obtain a butanol fraction (Bu) of Songmax extract.

4) Preparation of water fraction (W) of Songmae extract

The water layer remaining in Example 4-3 was concentrated under reduced pressure to obtain a water fraction (W) of Songmax extract.

Example  5: Songak extract and Hojo Chang  Preparation of a mixed composition comprising an extract

After shredding the shade and finely cut filaments, the filaments were repeatedly extracted with 1 liter of 80% ethanol, concentrated under reduced pressure at 40 ° C and dried under reduced pressure to obtain a callus root extract. The thus obtained Hojanggang extract was fractionated with ethyl acetate, butanol and water in the same manner as in Example 4 to prepare a Hojang root butanol fraction, which was mixed with the Hojak extract prepared in Example 4-1) The composition of Root Root Extract was prepared.

Experimental Example  One: The final glycation product ( advanced glycation end - products : AGEs ) Production inhibition effect experiment

Each of the compositions prepared in Example 1 and Example 2 was tested for the effect of inhibiting the final glycation endogenesis.

Protein was prepared by using bovine serum albumin (BSA, Sigma, USA) as an index and using aminoguanidine (AG) as an indicator to inhibit the formation of final glycosylation. And used as a control.

The protein source was prepared by mixing bovine serum albumin (BSA) in 50 mM phosphate buffer (pH 7.4) at a concentration of 10 mg / ml. The company used a mixture of 0.2 M fructose and 0.2 M glucose. Aminoguanidine (55.505 占 퐂 / ml, 74.75 占 퐂 / ml and 92.5 占 퐂 / ml) as the positive control and COPH (25 占 퐂 / ml, 50 占 퐂 / ml and 70 占 퐂 / After dissolving, 15% tween 80 was added. The total DMSO content was 0.2%. This was added to the mixture of bovine serum albumin and sugar and incubated at 37 ° C for 7 days. At this time, 0.02% sodium azide was added as an anti-bacterial agent. Bovine serum albumin and sugar mixture were used as controls. Blank of each test group and control group was not cultured after each preparation, and all of the cultures were prepared in triplicate to avoid the maximum error. After 7 days of culture, the content of the final glycation products produced in each culture was analyzed. The amount of the final saccharification product was measured by a microplate reader (Excitation: 350 nm, Emission; 450 nm), and the production inhibitory effect was calculated by the following formula (1). The results are shown in Table 1 below.

[Equation 1]

division Concentration (/ / ml) Inhibitory effect (%) IC ₅₀ ([mu] g / ml) COPH (Ivy leaf extract + Rhodiola extract) 25 35.53 + - 4.48 46.05 + 3.02 50 43.06 ± 2.82 70 51.08 + - 0.79 COPH + grape seed 1 (COPH: grape seed = 1: 1) 2.5 15.12 ± 1.20 7.29 + - 0.10 5 42.02 + - 1.28 10 65.10 + - 0.36 COPH + grape seed 2 (COPH + grape seed = 2: 1) 2.5 17.09 + - 4.48 18.80 + 0.07 5 39.15 + 1.32 10 60.85 +/- 0.76 COPH + grape seed 3 (COPH + grape seed = 3: 1) 2.5 18.03 ± 3.91 21.27 ± 0.58 5 39.15 + 1.32 10 60.85 +/- 0.76 COPH + Ginkgo leaf (COPH: Ginkgo leaf = 3: 1) 2.5 25.09 + 0.06 8.15 + 0.14 50 43.98 ± 1.56 10 58.49 + 1.30 COPH + Puerariae 1 (COPH: Puerariae = 1: 1) 10 9.41 ± 0.33 47.78 ± 1.24 25 23.73 ± 1.57 50 52.98 ± 2.06 COPH + Puerariae 2 (COPH: Puerariae = 3: 1) 25 22.02 ± 2.13 73.53 + - 0.79 50 33.28 ± 1.86 75 52.13 + - 0.49 COPH + Criterion 1 (COPH: Criterion = 1: 1) 10 19.54 + - 0.69 39.37 ± 0.60 25 31.18 + - 0.34 50 62.85 + - 0.98 COPH + Culprit 2 (COPH: Culprit = 2: 1) 10 21.80 ± 2.20 46.08 ± 1.22 25 29.94 + 1.57 50 54.21 + - 0.93 COPH + Culprit 3 (COPH: Culprit = 3: 1) 75 48.39 ± 0.39 77.79 ± 1.24 100 56.32 + - 0.23 125 57.76 ± 0.15 COPH + Rahul 1 (COPH: Rahul 1 = 1) 5 3.19 ± 1.25 21.50 ± 0.23 10 26.22 + -0.94 25 57.61 + - 0.76 COPH + Rahul 2 (COPH: Rahul 2 = 1) 10 7.57 ± 0.26 42.58 ± 1.02 25 29.50 ± 1.10 50 58.78 ± 1.75 COPH + bilberry 1 (COPH: bilberry = 1: 1) 10 18.37 + - 0.82 48.13 + - 1.12 25 32.85 + 0.66 50 50.92 + - 0.91 COPH + bilberry 2 (COPH: bilberry = 2: 1) 10 13.62 ± 0.09 51.69 ± 0.21 25 20.08 ± 0.53 50 50.18 + 0.09 COPH + bilberry 3 (COPH: bilberry = 3: 1) 10 12.26 ± 0.37 42.01 ± 0.29 25 33.49 + - 0.77 50 58.01 + - 0.27 COPH + bilberry 4 (COPH: bilberry = 1: 3) 5 23.85 ± 1.36 25.19 + - 0.67 10 28.66 ± 0.51 25 50.05 + - 0.70 Aminoguanidine HCl (positive control) 55.5 43.92 + - 0.37 76.47 + - 4.81 74.8 50.86 ± 2.17 92.5 54.91 + - 0.75

As shown in Table 1 above, it was confirmed that COPH, which is a composition containing ivy leaf extract and Rhodiola extract, is twice as effective as the aminoguanidine as a positive control, to inhibit the formation of final glycation end products.

In addition, a composition comprising Ivy leaf extract and Rhodiola extract and a composition prepared by adding grape seed, ginkgo leaf, gruel, lucerum, rhubarb, or bilberry extract to COPH is also excellent in the ratio of 2 to 10 times higher than that of aminoguanidine Inhibited the production of glycation products.

Experimental Example  2: STZ - Diabetes mellitus in an induced diabetic animal model (type 1 diabetes) Retinal edema  And Nephrotic  Effect analysis

In order to examine the effect of the composition prepared in Example 1 on diabetic retinopathy and nephropathy, the composition of Example 1 was orally administered to an STZ-induced mouse model of a type 1 diabetic animal, And the efficacy was confirmed.

1) Experimental animals

Male, 6-week-old SD rats were purchased from Orient Biotech Co., Ltd. and used for 1 week. For the induction of diabetes, STZ 60 mg / kg was injected into the rats by intraperitoneal injection. One week later, the blood glucose was checked and the individuals with blood glucose levels above 350 ㎎ / ㎗ were selected and separated into diabetic group and drug group. Respectively. The experimental group was divided into two groups: normal group (NOR), diabetic induction group (DM), the composition COPH 25 mg / kg / day administration group of Example 1 (COPH-25) and the composition COPH 50 mg / COPH-50). The composition COPH prepared in Example 1 was suspended in 0.5% carboxymethylcellulose (CMC) and administered orally for 3 weeks in each group.

2) Blood-retinal barrier breakage

To investigate the effect of blood retinal barrier damage (retinal edema), retinal vasculature was analyzed by weekly diabetic retinopathy for 3 weeks.

Eight diabetes-induced animal models were autopsied randomly in each group per week. After 10 mg / kg zolazepam (Zoletil, Virbac, France) and 10 mg / kg xylazine hydrochloride (Rumpun, Bayer, Germany) were anesthetized by intraperitoneal injection, the thoracic cavity was opened to obtain a heart, which was dissolved in 1 ml of sterile PBS mg / ml fluorescein-dextran (molecular weight 2 × 10 ⁶ ) and 1 mg / ml hochest 33342 (Simga, USA) were injected into the left ventricle. After 5 minutes, the eyeball was removed and the retina was separated from eyecup. The separated retina was placed on a slide and mounted with an aqueous mounting medium. After sufficiently dried, it was observed under a fluorescence microscope. For quantitative analysis, blood was collected from the heart 5 min after injection into the left ventricle of fluorescein-dextran (molecular weight 2 × 10 ⁶ ), perfused with sterile PBS to remove residual fluorescein-dextran in the blood vessel, Respectively. The separated retinas were homogenized in 100 μl of PBS, centrifuged, and the amount of FITC-dextran was measured using a spectrofluorophotometer. In addition, the amount of FITC-dextran was also measured in the previously collected blood, and the fluorescence value of the retina was corrected to quantify the tracer outflow. The results are shown in Fig.

As shown in FIG. 2, in the normal group (NOR), there was no phenomenon that the fluorescent substance injected into the blood vessels flowed out of the retinal blood vessels. However, in the diabetic induction group (DM) The amount of fluorescence emitted out was increased, and retinal tissue was observed brightly. On the other hand, in the group administered with the composition prepared in Example 1, COPH, the fluorescence flux was significantly inhibited in a concentration-dependent manner (FIG. In addition, the quantitative analysis also showed that the amount of the fluorescent substance in the retina tissue due to the blood vessel efflux was decreased in a dose-dependent manner in the group administered with the composition prepared in Example 1 (FIG. 2B).

3) Analysis of change of new functional index (effect on nephrotic syndrome)

To confirm the effect on nephrotic syndrome, proteinuria, final glycation products (AGEs) urine output, and oxidative stress marker 8-OHdG urine were analyzed.

Each week, urine was collected for each group and the supernatant was used for the experiment after spin-down to remove the impurities contained in urine. Protein concentration in the urine was measured by urine removal from the impurities. The protein concentration was quantitated by the Bradford method using Bio-Rad kit (Bio-Rad Laboratories Inc, USA).

Albumin urine output, final glycated products (AGEs) urine output, oxidative stress markers 8-OHdG urine output and synaptopodin urine output were measured by ELISA. First, 100 μl of urine was dispensed into a 96-well plate and cultured at 37 ° C for 2 hours for coating. Then, the cells were washed 3 times with PBS, incubated at 37 ° C for 40 minutes, washed, and 100 μl of each antibody (albumin, AGEs, 8-OHdG, Synaptopodin) (1: 2000 dilution) And reacted for 2 hours. After the reaction, each well was washed three times with PBS and 100 μl of Horseradish peroxidase-conjugated second antibody (1: 2000 dilution) was added and reacted. Thereafter, the cells were washed three times and developed with TMB (3,3 ', 5,5'-tetramethylbenzidine), and the reaction was stopped by addition of 0.5NH ₂ SO ₄ and the absorbance at 450 nm was measured. The measurement results are shown in Fig.

As shown in FIG. 3, in all the diabetic groups (DM), all of the three renal function indexes were significantly increased compared to the normal group (NOR) at 6 weeks of diabetes induction. On the other hand, the composition of the composition of Example 1, which was administered with COPH, showed a tendency to decrease in a concentration-dependent manner. This means that the COPH of Example 1 is effective for preventing diabetic nephropathy.

Experimental Example  3: db / db  Analysis of Diabetic Eye Disease Effect on Animal Model of Mouse (Type 2 Diabetes)

To examine the effect of the composition prepared in Example 1 and Example 3 on diabetic eye disease, COPH (ivory leaf extract + Rhodiola extract) of Example 1 was administered to a db / db mouse as a type 2 diabetic animal model, And COPH + MET (Ivy leaf extract + HuangYin extract + metformin) of Example 3 were orally administered. Experimental animals were purchased with male db / db mice at 6 weeks of age from SLC Japan and used for 1 week. The groups were randomly divided into groups with similar mean blood glucose and body weight before administration of the drug, and feed and drinking water were fed free. (COPH-25), 50 mg / kg of COPH (COPH-50) and 50 mg / kg of COPH were administered to the experimental group. (COPH-50) + (MET-350), and the drug was orally administered once daily for 12 weeks in each group. The embryos were fasted for 16 hours before the day of autopsy, and the organs were stored at -80 ° C.

1) Inhibitory effect of blood-retinal barrier breakage

If hyperglycemia persists, the blood retinal barrier is damaged due to abnormal function of the retinal vessels in the eye. Thus, it was confirmed that COPH of Example 1 and COPH + MET of Example 3 were prevented from damage to the blood retinal barrier. Each mouse in each experimental group was anesthetized by intraperitoneal injection of pentobarbital sodium (25 mg / kg body weight). The heart was opened by opening the abdominal cavity and thoracic cavity, and 50 mg / ml fluorescein-dextran (2 mg / × 10 ⁶ molecular weight) was injected into the left ventricle. After 10 minutes, the eyeball was removed and the left eyeball was separated into eyecups. The detached retina was placed on a slide and mounted with an aquious mounting medium. After sufficiently drying, the sample was observed under a fluorescence microscope. The results are shown in FIG. In order to observe retinal vasculature at a high magnification, serum albumin, which is a substance existing in blood, was stained using retinal tissue. The results are shown in Fig.

As shown in FIG. 4, no fluorescence leakage was observed in the normal group (NOR), but in the diabetic group (DM), fluorescence was leaked out of the blood vessel due to damage of the blood retinal barrier in the majority of individuals, (NOR) than that of the normal group (NOR). On the other hand, in the metformin-treated group (MET), fluorescein leakage was decreased in comparison with the diabetic group (DM), and the overall brightness was decreased compared to the diabetic group (DM). In the COPH administration group (COPH) of Example 1, the brightness due to fluorescence efflux was decreased in a concentration-dependent manner. In particular, the fluorescence effusion phenomenon was significantly reduced in the high concentration COPH administration group. In addition, COPH + MET mixed group showed the best inhibitory effect.

In addition, as shown in Fig. 5, in the diabetic group (DM), albumin leaks around the blood vessels and a pattern (arrow) stained brown was observed. However, this phenomenon was not observed in the combination of high-dose COPH and COPH + MET.

2) Immunohistochemical staining analysis

Immunohistochemical staining analysis was performed to confirm the effect of intercellular compaction, impaired vaginal protein degradation, accumulation of end glycation products and inhibition of vascular endothelial growth factor production. At the time of autopsy, the eyeballs were removed and fixed in 10% neutralized formalin overnight, dehydrated, replaced three times with xylene, and embedded in paraffin. Embedded tissue blocks were cut into 4 μm thick slices and placed on slides. After removal of endogenous peroxidase activity, the slides were deparaffinized and hydrolyzed. The slides were incubated in 3% hydrogen peroxide solution for 10 minutes and washed three times with PBS containing 0.05% tween 20. After blocking with 5% casein to remove nonspecific reaction, primary antibody was diluted 1: 200 each and treated for 1 hour or overnight. After washing with PBS for 1 hour, labeled streptoavidin biotin (LSAB) kit (Dako, USA) was applied and developed with DAB and observed with optical microscope. For fluorescence staining, FITC-conjugated secondary antibodies were diluted 1: 200 each, reacted for 1 hour, stained with DAPI, and observed with a fluorescence microscope. The results of each analysis are shown in FIG. 6 to FIG.

(1) Inhibitory effect of intracellular tight junction protein damage

As shown in FIG. 6, in the case of diabetes mellitus (DM), it was confirmed that the connecting line such as occludin thread was broken at various places of the blood vessel (arrow). However, it was confirmed that occludin disappearance was suppressed in COPH group and COPH + MET mixed group.

(2) Inhibitory Effect of Matrix Metalloproteinase-2 (MMP2) Expression

As shown in FIG. 7, the expression of MMP2 is increased (arrow) in the diabetic group (DM), whereas the expression of MMP2 is suppressed in the COPH administration group and the COPH + MET administration group.

(3) Inhibitory effect on the final glycation end product in retinal blood vessels

As shown in FIG. 8, in the diabetic group (DM), the final glycation end product was produced about twice as much as that in the normal (NOR) group, but the inhibitory effect was significant in the COPH administration group and the COPH + MET administration group.

(4) Inhibitory effect of vascular endothelial growth factor (VEGF) production

In general, VEGF, a vascular endothelial growth factor, induces the formation of new blood vessels and increases the permeability of blood vessels. In addition, there is a function of inducing the production of a substrate degrading protein. Therefore, in order to confirm that the effect of inhibiting vascular permeability of COPH and COPH + MET of the Example by inhibiting the production of vascular endothelial factor, VEGF staining analysis was performed and confirmed by ELISA. At this time, the ELISA was performed by homogenizing the retinas stored at -70 ° C with a homogenization buffer (pH 7.6), quantifying the proteins using the lowry principle, and using a VFGF ELISA kit (R & D system, USA) Respectively. The results are shown in Fig.

As shown in FIG. 9, the production of VEGF in the retina tissues was markedly increased in the diabetic group (DM), whereas the expression of VEGF was significantly inhibited in the COPH group and the metformin group (MET) . In addition, the VEGF concentration in the retinal tissues was confirmed by ELISA, and the production of VEGF was inhibited in a concentration dependent manner in the COPH administration group (COPH), and it was confirmed that VEGF production inhibiting activity was also excellent in the COPH + MET combination administration group.

3) Analysis of inhibitory effect on acellular capillary formation

One of the early symptoms of diabetic retinopathy is the pericyte nucleus, which is a peripheral cell caused by the non-metaplastic capillary formation. Therefore, a test for confirming the inhibitory effect of non-cellulosic capillary formation was carried out. The retina was extracted from the eyeball, washed with running water, and then incubated at 37 ° C for 1 hour in 3% trypsin. The digested retinas were transferred to PBS and the internal membrane (membrane of organelles present in the cells) was removed. The vascular frame was separated from the retina background using a glass rod, placed on a slide, and dried. PAS staining and hematoxylin staining revealed cell wall and nucleus changes. The results are shown in Fig.

As shown in FIG. 10, the number of non-metaplastic capillaries in each group was increased by about 5 times in the diabetic group (DM), whereas in the COPH administration group and the COPH + MET administration group, The formation of the vascular capillary was inhibited.

5) TUNEL staining analysis

Diabetic retinopathy can cause blindness in many ways, but optic nerve loss is one of them. It is generally known that loss of nerve by hyperglycemia is caused by apoptosis. Therefore, we performed TUNEL staining analysis to confirm cell death using retinal section. Tissues were hydrated by deparaffinization and treated with proteinase K at 20 ㎍ / ㎖ in PBS for 15 min at 37 ℃ and then washed again with PBS. TUNEL reaction mixture (Insitu cell death detection kit, AP, Roche, Germany) was reacted at 37 ° C for 1 hour and then observed with a microscope. The results are shown in Fig.

As shown in FIG. 11, in the diabetic group (DM), a strong fluorescence reaction (arrow) was observed in the nucleus of the ganglion cell, the innermost nerve cell in retinal tissue, whereas in the COPH administration group The number of TUNEL - stained retinal ganglion cells decreased in a dose - dependent manner and was significantly inhibited in COPH + MET mixed group.

6) Oxidative Stress Inhibition Efficacy Analysis

Oxidative stress is considered to be one of the major causes of diabetic retinopathy. Active oxygen (ROS) or active nitric oxide (RNS) induced by hyperglycemia causes damage to various cells in the retina. Therefore, the inhibitory effect of 8-OHdG, a marker of ROS, and nitrotyrosine, an RNS marker, was examined using retinal sections. The results are shown in Fig.

As shown in FIG. 12, it was confirmed that 8-OHdG and nitrotyrosine were all expressed in most cells including retinal ganglion cells present in the retina tissue in the diabetic group (DM). On the other hand, the COPH-treated group showed a decrease in the number of cells stained in a concentration-dependent manner and the most remarkably inhibited effect was observed in the COPH + MET mixed group.

Experimental Example  4: db / db  Inhibitory Effect of Diabetic Neuropathy on Animal Model of Mouse (Type 2 Diabetes)

The nerve conduction retardation was measured in order to examine the effect of the mixed composition (COPH) of the extract of Ivy leaf and Rhodiola extract prepared in Example 1 on neuropathy in an animal model of diabetic induction.

1) Experimental animals

Experimental animals were purchased from SLC Japan at 6 weeks of age in a male db / db mouse (type 2 diabetic animal model) and used for 1 week. After one week, blood glucose levels were measured and individuals with a body weight of more than 350 mg / ㎗ were randomly divided into groups. Feed and drinking water were fed free. The composition of Example 1, COPH, was suspended in 0.5% carboxymethylcellulose (CMC) and orally administered daily for 6 weeks. The experimental group was divided into the normal group (NOR), the diabetic group (DM), the composition of Example 1 (25 mg / kg / day of COPH) (COPH-25) and the composition of Example 1 of 50 mg / Treated group (COPH-50). Body weight, negative and feed intake were measured once a week.

2) Mortor neuron conduction velocity (MNCV)

Nerve conduction studies were performed for the analysis of delayed nerve conduction in diabetic peripheral. The mouse of Experimental Example 4-1) was anesthetized with isoflurane and fixed on a 37 ° C hot plate to maintain body temperature, and then measured in the caudal nerve using a nerve conduction analyzer (AD Intrument, Australia). At the tail base, electrical stimulation was applied using a needle electrode, and the action potential was recorded at a distance of 5 cm. The distance between the active electrode and the reference electrode was 1 cm. The stimulation time was 0.1 msec, the filtration frequency was 10 Hz to 10 kHz, the recording sensitivity was 5 mV / div, and the recording speed was 1 ms / div. A total of 10 measurements were made to calculate the mean value. The results are shown in Fig.

As shown in FIG. 13, the nerve conduction velocity was significantly lower in the DM group than in the normal group (NOR) after 6 weeks of diabetes induction. However, in the COPH-administered group of Example 1 of the present invention, the decrease in the nerve conduction velocity was improved in a concentration-dependent manner, and COPH-50, which is a group administered with COPH 50 mg / kg / day, showed a significant improvement in nerve conduction velocity. This means that the mixed composition of the above-mentioned Ivy leaf (Songak leaf) extract and Huanghui extract of the present invention is effective for the prevention of neuropathy caused by diabetes.

Experimental Example  5: In the zebrafish Antidiabetic complication  Effect analysis

(HR), the ethylacetate fraction (EA), the butanol fraction (Bu), the water fraction (W) and the sesame extract (W) of the Songmae ethanol extract prepared in Examples 4 and 5, In order to confirm the antidiabetic complication effect of the mixed composition (HR + Hojangun) of Schizandra chinensis extract, the vitellogenesis change analysis was carried out using zebrafish embryo.

1) Manufacture of zebrafish embryos and drug treatment

Zebrafish is one of the vertebrate animals that has relatively short experimental period and relatively low price compared to rodents such as mice. The zebrafish embryos were transfected with transgenic zebrafish (Tg (kdr: EGFP)) male and female embryos expressing green fluorescence protein specifically in vascular endothelial cells.

At 24 hours after fertilization, the embryos expressing the fluorescence were selected, and they were divided into 24 well plates and hyperglycemic environment was induced with 30 mM glucose. Each of the compositions prepared in Example 4 and Example 5 was diluted together with glucose solution. The experimental group was divided into two groups: normal group (NOR), hyperglycemic group (HG), 0.5 ㎍ / ㎖ treated group of HRP (0.5), 1 ㎍ / (EA (5)) treated with 1 ㎍ / ㎖ of ethyl acetate fraction, 5 ㎍ / ㎖ of ethyl acetate fraction of Songmax extract, (Bu (1)), butanol fraction (5 ㎍ / ㎖) (Bu (5)) of Seongak Extract, 1 ㎍ / ㎖ of water fraction of Songmax extract 1 ㎍ / ㎖ treatment group (0.5 ㎍ / ㎖), and 1 ㎍ / ㎖ treatment group (5 ㎍ / ㎖) (HR + Hojang (1)), a mixture of Songa extract and Hojanggang extract (0.5 ㎍ / ㎖), and 1 ㎍ / ).

2) Analysis of vitreous vascular changes

After 5 days of treatment under hyperglycemic induction conditions, they were fixed with 4% paraformaldehyde for one day. The changes of vitreous blood vessels were analyzed under fluorescent microscope by separating the lens from the fixed body. The results are shown in Fig. 14 and Fig.

As shown in Fig. 14, the hyperglycemia-inducing group (HG) expanded blood vessels as compared with the normal group (NOR). However, blood vessel enlargement due to hyperglycemia was significantly inhibited in the group treated with the sesame extract of Example 4 (1 ㎍ / ㎖ or 5 ㎍ / ㎖). Especially, when 5 ㎍ / ㎖ of Songak extract was treated, (NOR) vessels. In addition, ethyl acetate fraction (Bu) and butanol fraction (Bu) of sesame extract significantly inhibited hyperglycemia induced vitreous retinal vasodilatation at all experimental concentrations. Especially, 5 ㎍ / ㎖ of ethyl acetate fraction of Songak extract was treated (NOR) in the control group.

In addition, as shown in FIG. 15, it was confirmed that the diameter of the vitreous retina was maintained similar to that of the normal group (NOR) even when the composition of the sesame extract and the callus root extract of Example 5 was treated.

Experimental Example  6: VEGF  Inhibitory Effect of Retinal Blood Barrier Collapse on Induced Animal Model

In order to confirm the preventive or therapeutic effect of proliferative retinopathy caused by angioedema of the mixed composition (COPH) of the ivy leaf extract and the Rhodiola extract of Example 1, VEGF was injected into the eyeball to induce disease, and at the same time, (COPH) was applied to the vitreous body of the eye to evaluate the efficacy of preventing the blood-retinal barrier disruption, one of the early symptoms of diabetic retinopathy. When the blood barrier collapses, proteins, red blood cells, and the like disappear from the blood vessels in the retina, and then progress to the macula of the retina and further to proliferative retinopathy. In the case of the lower limb,

1) Experimental animals

Seven male male SD rats were purchased from the Orient and used for 1 week. Feed and drinking water fasted freely. To induce collapse of blood retinal barrier, 200 ng of Rat VEGF protein (Vascular endothelial growth factor, R & D research, USA) was dissolved in sterile physiological saline and 5 μl was injected into the left eye using a hamilton syringe (Hamilton, USA). In the right eye, only physiological saline was administered as a control. COPH groups were dissolved in 0.5 DMSO solution, and 5 μl of the solution was administered to both the left and right eye so that the final concentration in the eyeball was 1 μg / ml, 5 μg / ml and 10 μg / ml. In general, since the amount of the vitreous body in the eye is approximately 50 to 55 μl, the final concentration is adjusted by administering 12 μg / ml, 60 μg / ml and 120 μg / ml of the drug concentrated to 12 times the final concentration in the eye.

2) Blood retinal barrier damage analysis

Twenty - four hours after administration of the drug, each rat was anesthetized by intraperitoneal injection of pentobarbital sodium (25 mg / kg body weight). The heart was opened by opening the abdominal cavity and thoracic cavity, and 50 mg / ml fluorescein-dextran (2 × 10 ⁶ molecular weight) prepared by dissolving in 1 ml / ml sterile PBS was injected into the left ventricle. After 10 minutes, the eyeball was removed and in the left eye, the retina was separated from eyecup. The separated retina was placed on a slide and mounted with an aqueous mounting medium. After sufficiently dried, it was observed with a fluorescence microscope. For quantitative analysis, blood samples were collected 10 min after injecting 50 ㎎ / ㎖ fluorescein-dextran (2 × 10 ⁶ molecular weight) into the left ventricle and analyzed with fluorescein-dextran at 60 ml / min using physiological saline And the extracted retina was separated from the eyeball. The separated retinas were weighed, homogenized in 50 μl of PBS solution, finely pulverized, centrifuged and the supernatant was measured for fluorescence with an ELISA reader. The results are shown in Fig. 16 and Fig.

As shown in FIG. 16, fluorescein was injected into the blood vessels of the retina to observe the extracellular leakage of the ciliary material due to damage of the blood retinal barrier. As a result, no fluorescence leakage was observed in the normal group (NOR) In the VEGF intra-ocular administration group, fluorescence was observed to leak out of the blood vessel due to blood retinal barrier damage in all individuals. On the other hand, in the COPH administration group, fluorescence efflux due to VEGF was reduced, and fluorescence brightness was decreased in a concentration dependent manner.

In addition, as shown in FIG. 17, the quantitative analysis also revealed that the VEGF-treated group emitted 5 times or more of the fluorescent substance out of the blood vessels compared to the normal group (NOR), while the COPH administration group decreased in a dose- At the concentration of 10 ㎍ / ㎖, it was confirmed that the fluorescence leakage due to damage of the blood retinal barrier was inhibited to the same extent as that of the normal group.

Claims (15)

A pharmaceutical composition for prevention or treatment of diabetic complications or angioedema comprising an extract of Ivy leaf and Rhodiola as an active ingredient,
The diabetic complication is any one selected from the group consisting of diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic cataract, diabetic arteriosclerosis, or diabetic complications resulting from the production of a final glycated product,
Wherein said angioedema is macular edema.
The composition according to claim 1, wherein the composition is prepared by mixing ivy leaf extract and rhododendron extract at a weight ratio of 10: 1 to 1:10, or mixing ivy leaves and phloxy at a weight ratio of 10: 1 to 1:10 And extracting the composition.
The pharmaceutical composition according to claim 1, wherein the composition further comprises an extract of Hojangun, Rhubarb, Puerariae, Ginkgo biloba, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe.
2. The pharmaceutical composition according to claim 1, wherein the composition further comprises metformin.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the ivy leaf extract and Rhodiola extract are obtained by extracting ivy leaves and Rhodiola with water, methanol, ethanol, butanol or a mixed solvent thereof.
[Claim 7] The pharmaceutical composition according to claim 5, wherein the extraction is a normal temperature extraction, a hot water extraction, a cold extraction, a reflux cooling extraction, an ultrasonic extraction, a supercritical or a steam extraction.
delete delete A health functional food composition for preventing or ameliorating diabetic complications or angioedema comprising a mixed extract of ivy leaves and Rhodiola as an active ingredient,
The diabetic complication is any one selected from the group consisting of diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic cataract, diabetic arteriosclerosis, or diabetic complications resulting from the production of a final glycated product,
Wherein said angioedema is macular edema.
[Claim 11] The composition according to claim 9, wherein the composition is prepared by mixing ivy leaf extract and rhododendron extract at a weight ratio of 10: 1 to 1:10, or mixing ivy leaf and phlox at a weight ratio of 10: 1 to 1:10 And extracting the composition.
[Claim 11] The composition according to claim 9, wherein the composition further comprises Hojangun, Rhubarb, Puerariae, Ginkgo biloba, Cucumber, Blueberry, Bilberry, Bokbunja, Licorice, Grape Seed or Aloe.
[Claim 11] The health functional food composition according to claim 9, wherein the ivy leaf extract and the Rhodiola extract are obtained by extracting ivy leaves and Rhodiola with water, methanol, ethanol, butanol or a mixed solvent thereof.
The health functional food composition according to claim 12, wherein the extraction is at room temperature extraction, hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction, supercritical or steam extraction.
delete delete

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